Cytokine-based Immunotherapy Research Articles

172. Early Considerations in Schizophrenia Immunotherapy

Abstract Overall Abstract: Overview: A pathophysiological role for inflammation in schizophrenia has been an enduring finding. Several trials have found that adjunctive treatment with non-steroidal anti-inflammatory drugs (NSAIDs) are associated with improvement in psychopathology and cognition in schizophrenia. These findings provide important empirical support for a pathophysiological role for inflammation in some patients with schizophrenia. To date, two small studies of cytokine-based immunotherapy in schizophrenia have been published. Two major potential advantages of monoclonal antibody immunotherapy over NSAIDs or other anti-inflammatory agents are that monoclonal antibodies are more potent than other anti-inflammatory agents and do not have any off-target (i.e., nonimmune) effects, thereby directly testing the role of neuroinflammation in the pathophysiology of schizophrenia. Description: This workshop will discuss early experiences, including study design and considerations, of ongoing trials of monoclonal antibody immunotherapy in schizophrenia. First, Dr. Oliver Howes will present new PET imaging and preclinical data implicating microglial activation in the pathophysiology of schizophrenia. A randomized controlled trial (RCT) of natalizumab in schizophrenia, which reduces microglial overactivity by targeting the cell adhesion molecule α4-integrin, is ongoing. Dr. Cyndi Shannon Weickert will then discuss new data suggesting inflammation is associated with altered blood–brain barrier function in schizophrenia, as well as cognitive and neuropathological changes. Next, Dr. Thomas Weickert will discuss an ongoing RCT of adjunctive canakinumab (anti-IL-1β) in schizophrenia. Finally, Dr. Brian Miller will discuss two ongoing RCTs of adjunctive anti-IL-6 therapy (tocilizumab, an IL-6 receptor antagonist, and siltuximab, an IL-6 antagonist) for cognitive impairment in schizophrenia. Lastly, Professor Norbert Mueller, the workshop moderator will summarize his perception of the current state of the field of investigations of immunotherapy in schizophrenia. Attendees will learn about the strengths and limitations of current research in the field and will have the opportunity to engage in a (hopefully lively) dialogue about the future directions of work in this area.

Unveil the mysterious mask of cytokine-based immunotherapy for melanoma

Melanoma is the leading cause of death among all skin cancers and its incidence continues to rise rapidly worldwide in the past decades. The available treatment options for melanoma remain limited despite extensive clinical research. Melanoma is an immunogenic tumor and great advances in immunology in recent decades allow for the development of immunotherapeutic agents against melanoma. In recent years, immunotherapy utilizing cytokines has been particularly successful in certain cancers and holds promise for patients with advanced melanoma. In this review, an overview of the current status and emerging perspectives on cytokine immunotherapy for melanoma are discussed in details. Such a study will be helpful to unveil the mysterious mask of cytokine-based immunotherapy for melanoma.

The SCCHN drug market.

Two PD-1 directed checkpoint inhibitors have recently been approved for squamous cell carcinoma of the head and neck (SCCHN). Further checkpoint inhibitors, as well as other molecularly targeted agents and cytokine-based immunotherapies, are currently in the late-stage pipeline and are poised to change the treatment paradigm for SCCHN.

Immunotherapy of melanoma

Immunotherapy for advanced melanoma has progressed dramatically in the last five years with the approval of immune checkpoint inhibitors targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Inhibition of these targets can break cancer-immune tolerance and result in durable objective responses with significantly improved tolerability over cytokine-based immunotherapy. Ipilimumab is an inhibitor of CTLA-4 and the first-in-class immune checkpoint inhibitor to demonstrate an improvement in overall survival in melanoma. Pembrolizumab and nivolumab target PD-1 and have improved single agent activity and tolerability in comparison to ipilimumab. The combination of nivolumab and ipilimumab results in even better response rates, reductions in tumor volume and progression free survival but at the expense of considerable autoimmune effects. Autoimmune side-effects and non-standard response kinetics represent a new challenge associated with cancer therapies that practitioners will have to become more familiar with as checkpoint inhibitors increasingly become part of mainstream oncological practice. Ongoing areas of investigation include drug development against novel immune targets; alternative treatment modalities, such as genetically modified oncolytic viruses; optimization of immunotherapy combination strategies; and the identification of reliable biomarkers to better guide treatment selection.

The promising alliance of anti-cancer electrochemotherapy with immunotherapy

Anti-tumor electrochemotherapy, which consists in increasing anti-cancer drug uptake by means of electroporation, is now implanted in about 140 cancer treatment centers in Europe. Its use is supported by the English National Institute for Health and Care Excellence for the palliative treatment of skin metastases, and about 13,000 cancer patients were treated by this technology by the end of 2015. Efforts are now focused on turning this local anti-tumor treatment into a systemic one. Electrogenetherapy, that is the electroporation-mediated transfer of therapeutic genes, is currently under clinical evaluation and has brought excitement to enlarge the anti-cancer armamentarium. Among the promising electrogenetherapy strategies, DNA vaccination and cytokine-based immunotherapy aim at stimulating anti-tumor immunity. We review here the interests and state of development of both electrochemotherapy and electrogenetherapy. We then emphasize the potent beneficial outcome of the combination of electrochemotherapy with immunotherapy, such as immune checkpoint inhibitors or strategies based on electrogenetherapy, to simultaneously achieve excellent local debulking anti-tumor responses and systemic anti-metastatic effects.

Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells.In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15).FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05).Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15).C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor.Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. DisclosuresNg:Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.

Associations of Fc gamma receptor (FcgR2a, FcgR3a and FcgR2c) genotype with outcome in metastatic renal cell carcinoma (mRCC) patients receiving high dose interleukin 2 (HD-IL2)

HD-IL2 was given to patients with mRCC in a prospective trial (ClinicalTrials.gov ID: NCT00554515) that demonstrated an overall response rate of 25%. To identify predictors of response, we genotyped patients for single nucleotide polymorphisms (SNPs) in FcgR genes: FcgR2a, FcgR3a, and FcgR2c. These FcgRs are variably expressed on immune cells, and bind to the IgG portion of antibodies, triggering activation. FcgR2a [SNP = histidine/arginine (H/R); expressed on neutrophils, monocytes-macrophages and antigen-presenting cells (APCs)] and FcgR3a [SNP = valine/phenylalanine (V/F); expressed on NK cells and some APCs] impact patient response to immunotherapy [mainly monoclonal antibody-based (mAb) therapies] in several malignancies, as both FcgR2a-H and FcgR3a-V have higher antibody binding affinity than FcgR2a-R or FcgR3a-F, respectively. FcgR2c, also expressed on NK cells, has a SNP that regulates its expression [C nucleotide = expression; T nucleotide = non-expression (C/T)]. About 20-40% of individuals express FcgR2c; little is known about the role of FcgR2c expression in cytokine-based immunotherapy. We found associations of FcgR genotypes with patient response to HD-IL2. Dual combination analyses of FcgR3a with FcgR2a genotypes revealed significantly improved %Tumor Shrinkage in patients with either FcgR3a-VV and/or FcgR2a-HH (n=34) as compared to those patients genotyped as FcgR3a-VF or FF with FcgR2a-HR or RR (n=70) (p=0.047). For analyses including both FcgR3a and FcgR2c genotypes, significantly improved OS was seen in those with ≥2 alleles of either FcgR3a-V and/or FcgR2c-C (n=27) vs. those with < 2 alleles of either FcgR3a-V and/or FcgR2c-C (n=79) (p=0.013). We further considered combinations that included the genotypes of all 3 genes; we identified 42 patients with “favorable” genotypes and 64 with “unfavorable”. We saw significant improvement in the %Tumor shrinkage (p = 0.033) and a trend for improvement in OS (p = 0.071) in the “favorable genotype” group. As reported previously by others, some cancer patients make endogenous anti-tumor Ab. The association of these “favorable” FcgR genotypes with outcome suggests there may be a beneficial interaction of “favorable” FcR genotypes with endogenous anti-tumor Ab. Favorable FcgRs may support the induction of in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). In addition, ADCP by APC with a favorable pattern of FcgRs, may be playing a role in enhancing induction of effective adaptive immunity, via augmented antigen presentation. Further analyses are needed to validate these exploratory findings and clarify the mechanisms by which these favorable FcgR genotypes are associated with improved outcome in these patients with mRCC treated with HD-IL2.

Immunotherapy Approaches in Cancer Treatment.

Tumor immune surveillance paradigm presumes that most pre-malignant cells or early malignant lesions can be eliminated (or at least controlled) by cells of the immune system. A critical feature that distinguishes advanced tumors from early neoplastic lesions is their capability to evade immune control. As a consequence, vast majority of clinically evident (advanced) tumors are poorly immunogenic. The principle goal of immunotherapy is thus a resurrection of the patient's inefficient or suppressed immune system so that it would once again become capable of launching sustained cytolytic attacks against tumor cells, which would ideally result in total and permanent eradication of cancer. Such activation of patient's anticancer immunity, however, can be achieved by strikingly different ways. This current review discusses diverse innovative immunotherapy approaches, which in the last 20 years achieved miraculous successes in the ever-lasting battle against cancer, including cytokine-based immunotherapy approaches, therapeutic monoclonal antibodies and their derivatives, cancer vaccines, and cell-based immunotherapy approaches.

IL-9 inhibits HTB-72 melanoma cell growth through upregulation of p21 and TRAIL.

IL-9 is a pleiotropic cytokine produced mainly by Th9 cells. IL-9 may have an anti-proliferative role in murine melanoma, however, its effect on human melanoma is unknown. We examined the effects of IL-9 on proliferation and apoptosis in four human melanoma cell lines, HTB-65, HTB-72, CRL-11147, and SK-Mel-5. Clonogenic assay, PCNA staining, Quick Cell Proliferation assay, TUNEL staining and caspase-3 activity assay were used to assess proliferation and apoptosis, as appropriate. We found that IL-9 decreased the percentage of colonies of HTB-72 and SK-Mel-5 cells but not that of HTB-65 or CRL-11147 cells. PCNA mRNA, PCNA+ cells, PCNA staining intensity, and the OD value of HTB-72 melanoma cells were consistently decreased in the present of IL-9. IL-9 also increased TUNEL+ cells and the relative caspase-3 activity in HTB-72 melanoma cells. We further investigated the possible molecular mechanisms using RT-PCR and immunohistochemical staining. The anti-proliferative effect of IL-9 on HTB-72 cells correlated with higher expression of anti-proliferative molecule p21. Its pro-apoptotic effect on HTB-72 cells correlated with higher expression of the pro-apoptotic molecule TRAIL. IL-9 inhibits melanoma HTB-72 cell growth by upregulation of p21 and TRAIL. Understanding the interactions between IL-9 and melanoma may help direct strategies for cytokine-based immunotherapy development.

Combination therapy of an IL-15 superagonist complex, ALT-803, and a tumor targeting monoclonal antibody promotes direct antitumor activity and protective vaccinal effect in a syngenic mouse melanoma model

Cytokine-based and antibody-targeted immunotherapies have both been important approaches in the treatment of malignant cancers. However, combinational therapies of cytokines and tumor-targeting antibodies remain to be further explored, especially in advanced solid tumors. In this study, C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with mouse melanoma targeting anti-gp75 monoclonal antibody (mAb), TA99, combined with interleukin (IL)-15 based superagonist ALT-803. This soluble protein complex consists of an IL-15 superagonist mutant (IL-15N72D) associated with an IL-15 receptor α Sushi domain - human IgG1 Fc fusion protein. Compared to native IL-15, ALT-803 possesses superior in vivo biologic activity for stimulating NK and CD8+ memory T cells. The combined ALT-803+TA99 therapy significantly exceeded either monotherapy in inhibiting melanoma tumor growth (p < 0.001) and prolonging survival (p < 0.01) of B16F10 tumor-bearing mice. Through immune cell depletion studies and immunophenotyping of peripheral cells as well as tumor-infiltrating leukocyte subsets, we found that ALT-803 enhances TA99-mediated antitumor immunity through activation of NK cells and expansion of the CD8+CD44high memory T cell arm. In contrast, CD4+ T cells were shown to play more of a suppressive role in the therapeutic effect of ALT-803+TA99, possibly through involvement of regulatory T cells or ALT-803-mediated induction of PD-L1 on CD4+ T cells in the periphery and tumor microenvironment. Addition of anti-PD-L1 mAb to ALT-803+TA99 therapy resulted in a further increase in antitumor activity against subcutaneous B16F10 tumors. Furthermore, tumor-bearing mice that survived due to ALT-803+TA99 combination therapy exhibited long term antitumor memory against B16F10 tumor cell rechallenge. Immune-depletion studies revealed that this antitumor memory was associated with CD4+ T cells, CD8+ T cells and NK cells. Our findings suggest a therapeutic opportunity for ALT-803 in combination with tumor-targeting antibodies to simultaneously augment targeted antitumor activities of therapeutic antibodies and induce a long-term vaccinal effect which will provide durable responses in the treated host.

Fever and the use of paracetamol during IL-2-based immunotherapy in metastatic melanoma.

Fever is frequently observed in conjunction with interleukin-2 (IL-2)-based immunotherapy. Traditionally, fever has been regarded as an undesirable side effect and treated with fever-lowering drugs. However, new insights in tumor immunology suggest that elevated temperature may facilitate a more effective antitumor immune response. The purpose of this retrospective study was to examine the potential role of the IL-2-induced fever in melanoma patients treated with or without paracetamol in two consecutive cohorts. One hundred and seventy-nine patients with metastatic melanoma treated with a modified decrescendo regimen of IL-2 and Interferon (IFN) between 2004 and 2010 were retrospectively studied. 87 patients treated before 2007 received paracetamol as part of the treatment schedule, and 92 patients treated after 2007 did not receive paracetamol routinely. Body temperature was analyzed as dichotomized and continuous variables and correlated to objective tumor response and overall survival using logistic regression and Cox proportional hazard analysis. Patients experiencing peak temperature of ≥ 39.5 °C had a median OS of 15.2 months compared to 8.7 months among patients with lower temperatures (P = 0.01). In the multivariate analysis, peak temperature of ≥ 39.5 °C (HR 0.53; P = 0.026) and high mean temperature (HR 0.56; P = 0.004) were independent prognostic factors for improved survival. We suggest high fever as a biomarker for improved survival in melanoma patients treated with IL-2/IFN. The routine use of fever-reducing drugs during immunotherapy can therefore be questioned. More studies are needed to evaluate the role of fever and the use of antipyretics during cytokine-based immunotherapy.

Concise review: engineering the fusion of cytokines for the modulation of immune cellular responses in cancer and autoimmune disorders.

As our understanding of the basic precepts of immunobiology continue to advance at a rapid pace, translating such discoveries into meaningful therapies for patients has proved challenging. This is especially apparent in the use of cytokine-based immunotherapies for cancer. Unanticipated and serious side effects, as well as low objective response rates seen in clinical trials, have dealt setbacks to the field. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and common γ-chain (γ-c) interleukins are cytokines that have been used as stand-alone immunotherapies with moderate success. Our group has found that the fusion of GM-CSF to members of γ-c interleukins results in the generation of novel proteins with unique signaling properties and unheralded biological effects. These fusion proteins, termed GIFT (GM-CSF interleukin fusion transgenes) fusokines, are the result of combining GM-CSF and a γ-c interleukin into a single, bifunctional polypeptide. In our experience, GIFT fusokines often confer immune cells with a gain of function that cannot be explained by the mere sum of their constituent moieties. They act as bispecific ligands, coupling activated GM-CSF and interleukin receptors together to drive unique downstream signaling events. The synergy that arises from these fusions has shown great promise in its ability to modulate the immune response and overcome maladaptive biological processes that underlie diseases such as cancer and autoimmune conditions. In this review, we discuss the ways in which the GIFT fusokines are able to alter the immune response, particularly in disease states, with a special emphasis on how these novel molecules may be translated into effective therapies in the clinical setting.

Cytokine therapy reverses NK cell anergy in MHC-deficient tumors.

Various cytokines have been evaluated as potential anticancer drugs; however, most cytokine trials have shown relatively low efficacy. Here, we found that treatments with IL-12 and IL-18 or with a mutant form of IL-2 (the "superkine" called H9) provided substantial therapeutic benefit for mice specifically bearing MHC class I-deficient tumors, but these treatments were ineffective for mice with matched MHC class I+ tumors. Cytokine efficacy was linked to the reversal of the anergic state of NK cells that specifically occurred in MHC class I-deficient tumors, but not MHC class I+ tumors. NK cell anergy was accompanied by impaired early signal transduction and was locally imparted by the presence of MHC class I-deficient tumor cells, even when such cells were a minor population in a tumor mixture. These results demonstrate that MHC class I-deficient tumor cells can escape from the immune response by functionally inactivating NK cells, and suggest cytokine-based immunotherapy as a potential strategy for MHC class I-deficient tumors. These results suggest that such cytokine therapies would be optimized by stratification of patients. Moreover, our results suggest that such treatments may be highly beneficial in the context of therapies to enhance NK cell functions in cancer patients.

The IL-2 cytokine family in cancer immunotherapy

The use of cytokines from the IL-2 family (also called the common γ chain cytokine family) such as interleukin (IL)-2, IL-7, IL-15, and IL-21 to activate the immune system of cancer patients is one of the most important areas of current cancer immunotherapy research. The infusion of IL-2 at low or high doses for multiple cycles in patients with metastatic melanoma and renal cell carcinoma was the first successful immunotherapy for cancer proving that the immune system could completely eradicate tumor cells under certain conditions. The initial clinical success observed in some IL-2-treated patients encouraged further efforts focused on developing and improving the application of other IL-2 family cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21) that have unique biological effects playing important roles in the development, proliferation, and function of specific subsets of lymphocytes at different stages of differentiation with some overlapping effects with IL-2. IL-7, IL-15, and IL-21, as well as mutant forms or variants of IL-2, are now also being actively pursued in the clinic with some measured early successes. In this review, we summarize the current knowledge on the biology of the IL-2 cytokine family focusing on IL-2, IL-15 and IL-21. We discuss the similarities and differences between the signaling pathways mediated by these cytokines and their immunomodulatory effects on different subsets of immune cells. Current clinical application of IL-2, IL-15 and IL-21 either as single agents or in combination with other biological agents and the limitation and potential drawbacks of these cytokines for cancer immunotherapy are also described. Lastly, we discuss the future direction of research on these cytokines, such as the development of new cytokine mutants and variants for improving cytokine-based immunotherapy through differential binding to specific receptor subunits.

Cytokine-based cancer immunotherapy impairs primary T cell immunity (VAC3P.942)

Abstract Cancer immunotherapy has made great strides towards becoming a viable alternative to traditional cancer treatment options. Under the premise of specificity and long lasting memory, immunotherapy is hypothesized to provide what traditional cancer therapies lack. However, despite success in tumor efficacy, the smoking gun of tumor-specific immunity has yet to be uncovered. Few studies to date have found correlations between tumor specific T cells and outcomes, particularly with stimulatory regimens. Here we show evidence that following cytokine based immunotherapy regimens, primary CD4 T cell responses towards mitogenic or cognate antigenic stimulation were greatly hampered. This lack of CD4 immunity was further exacerbated by acute thymic involution. Interestingly, the paralysis was confined to the CD4 T cell subset and was transient, resolving within 2 weeks of cessation of therapy. Paralyzed T cells significantly upregulated SOCS3 and failed to undergo proper STAT phosphorylation and CD25 upregulation following TCR engagement. The consequences of this paralysis were impaired vaccination and viral clearance, and also suggest impairment in the development of subsequent primary anti-tumor T cell responses. Taken together, these data suggest that cytokine-based immunotherapies may actually impair the development of immunity towards novel tumor antigens and may put patients at risk for opportunistic infections.

Pazopanib-the Latest First-Line Standard of Care Targeted Kinase Inhibitor for the Treatment of Advanced Renal Cell Carcinoma

Kidney cancer is the most lethal of the genitourinary malignancies. Prior to the advent of cytokine-based immunotherapy 25 years ago, surgery was the only viable treatment for advanced renal cell carcinoma (RCC). Adjuvant therapy with interferon and interleukin-2 revolutionized the treatment paradigm, although responders were too often uncommon. The mid-portion of the last decade saw born a new class of drugs aimed at the angiogenesis/VEGF pathway, common to most clear-cell RCC. The latest FDA-approved compound in this class has promised to yield equal or better first-line efficacy as its predecessors while, perhaps, providing better tolerability. Here we review the available data at present regarding pazopanib.

Soypeptide lunasin in cytokine immunotherapy for lymphoma

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Activation Of Natural Killer Cells By Soypeptide Lunasin and Cytokine: Implication In Cancer Immunotherapy For Lymphoma

IntroductionImmunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that chemotherapy-treated lymphoma patients have acquired deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNg production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer (NK) cells. Experimental DesignPBMCs of healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. ResultsAdding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The expression level of CD16 and granzyme B was increased in CD56-bright population of NK cells following stimulation with lunasin and cytokine. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNg production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines had higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. Moreover, lunasin augmented antibody-dependent cellular cytotoxicity (ADCC) of NK cells against anti-CD20 coated human B-lymphoma cell line. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. ConclusionWe have discovered a novel use of lunasin that exerts synergistic effects together with IL-12 or IL-2. This synergism leads to stronger NK-mediated anti-tumor activity, suggesting the potential clinical use of lunasin to augment the therapeutic responses in cytokine-based immunotherapy. Disclosures:No relevant conflicts of interest to declare.

An engineered immunotherapy (NKTR-214) with altered selectivity toward the IL2 receptor: Efficacy and tolerability in a murine tumor model.

3060 Background: Cytokine-based immunotherapy has been successful for the treatment of cancer, with potential for durable responses in multiple indications. One approach towards stimulating the immune system is to target the heterotrimeric interleukin2 receptor, IL2R. NKTR-214 uses polymer technology to alter receptor subunit selectivity to favor expansion of CD8+ memory effector T cells (CD8T) in the tumor over CD4+ regulatory T cells (Treg). In addition, polymer conjugation is designed to improve exposure and enhance tumor localization, significantly improving efficacy, modulating vascular leak syndrome (VLS) and allowing flexible dosing regimens. Methods: C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with NKTR-214 at a variety of doses (0.25-4.0 mg/kg) and schedules (q5dx3 to q14dx2). Mice treated with clinically validated IL2 were administered 3mg/kg, bidx5 for 2 cycles. Efficacy was measured by monitoring tumor volumes. Tolerability was evaluated by survival. VLS was measured by injection of Evans Blue dye followed by colorimetry in lungs. Tumor immunotyping, by flow cytometry. Results: Tumors from mice receiving NKTR-214 had a CD8/Treg ratio of over 1,000 versus 14 for IL2. NKTR-214 administered at 2 mg/kg, q9dx3 was identified as the optimal regimen and showed tumor growth delay of 26 days compared to 9 days for optimally dosed IL2. 90% of NKTR-214 treated mice tolerated treatment compared to 67% for IL2. VLS was completely resolved prior to administration of the next dose of NKTR-214, unlike IL2. NKTR-214 was well tolerated in rats at two schedules, at MTD. Conclusions: NKTR-214 is a highly differentiated cytokine with a new mechanism of action that may provide options for cancer immunotherapy. Polymer conjugation of a clinically validated cytokine alters the IL2R selectivity to favor expansion of tumor killing immune cells (CD8T) over regulatory immune cells (Treg) in the tumor. The conjugate is also designed to improve exposure and enhance tumor localization, offering more options of dose and schedule. The optimal dose and schedule is cytokine-sparing, provides substantial tumor growth delay, and reduces toxicity.

Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity

Local delivery systems that provide sustained, high concentrations of antitumor cytokines in the tumor microenvironment while minimizing systemic dissemination are needed to realize the potential of cytokine-based immunotherapies. Recently, co-formulations of cytokines with chitosan solutions have been shown to increase local cytokine retention and bioactivity. In particular, intratumoral (i.t.) injections of chitosan/IL-12 can eliminate established tumors and generate tumor-specific immune responses. In the present study, we explored the mechanisms by which chitosan potentiated IL-12's antitumor activity. The location of chitosan/IL-12 injection was found to be critical for optimal cytokine delivery. I.t. injections eliminated 9 of 10 MC38 adenocarcinomas while contralateral and peritumoral injections delayed tumor growth but could not eliminate tumors. Microdosing studies demonstrated that IL-12 depots, simulated through daily i.t. injections with IL-12 alone, were not as effective as weekly i.t. chitosan/IL-12. 50–75% of mice receiving daily IL-12 microdoses and 87.5% of mice receiving weekly chitosan/IL-12 were cured of MC38 tumors. Chitosan was found to increase IL-12-mediated leukocytic expansion in tumors and tumor-draining lymph nodes (TDLNs) by 40 and 100%, respectively. Immunophenotyping studies demonstrated that chitosan co-formulation amplified IL-12-induced increases in important effector populations, such as CD8+IFN-γ+ and NKT cells, in tumors and dendritic cell populations in TDLNs. Remarkable increases in Gr-1+CD11b+ tumor infiltrates were also observed in mice receiving chitosan or chitosan/IL-12. This population does not appear be suppressive and may facilitate the local antitumor response. Presented data suggest that chitosan-mediated depot formation and enhanced local cytokine retention is significantly, but not entirely, responsible for increased cytokine bioactivity.